H. pylori is the only bacterium to inhabit the normal human stomach. As a neutralophile only able to grow between pH 6.0 and 8.0, it has evolved several specialized acid adaptive mechanisms to allow it to thrive in the variable acidic milieu of the mammalian stomach. A vital means of elevation of periplasmic and cytoplasmic pH in acidic external pH is achieved via a pH-gated urea channel, Urel, in the inner membrane that allows urea access to intra-bacterial urease at acidic pH. NH3 generated internally increases both cytoplasmic and periplasmic pH, but not to levels found at neutral external pH when external pH is below 6.0. Other acid adaptive processes are also activated by the reduction in cytoplasmic pH and/or periplasmic pH. The specific aims are (a) fluorimetric cDNA microarray analysis of the complete genome of H. pylori to identify genes whose expression is up regulated in response to several different external pH values between pH 2.5 and 6.2 in the absence and presence of physiological concentrations of urea. Among these genes, those that are also able to regulate pHin will be investigated further. Their increase found on the micro-arrays will be confirmed by use of real time PCR. (b) the relative increase in their gene expression will be correlated with changes of the internal pH and inner membrane potential difference to determine the site of pH regulation of expression (c) to show that increased gene expression is associated with increased protein abundance and to also detect proteins whose levels may rise without an increase in gene transcription, changes in protein levels in response to acid will be assessed using pulse labeling, 2D gel electrophoresis and mass spectrometry. Western analysis and enzyme assay will also confirm increased levels of some of the pH regulatory genes and (d) to show that these pH regulatory processes have a role in acid adaptation, the effect of deletion of each of these genes on internal pH and on growth and survival of H. pylori in acid will be determined. These studies should provide further insight as to how H. pylori has evolved to colonize the human stomach. These studies may provide targets to allow eradication of this class 1 carcinogen without the use of general antibiotics.